TOTAL INTERNAL-REFLECTION MICROSCOPY - A QUANTITATIVE TOOL FOR THE MEASUREMENT OF COLLOIDAL FORCES

Total internal reflection microscopy (TIRM) is a new technique used to measure directly the mean potential of the interaction between a microscopic sphere and a glass plate. TIRM capitalizes on the properties of the evanescent wave produced by total internal reflection at the glass/liquid interface. When any portion of the sphere is present within the range of the evanescent wave, it will scatter light. The intensity of the scattered light decays exponentially with the distance separating the sphere and the plate. This provides a sensitive, instantaneous, and nonintrusive measure of the submicroscopic separation distance. A histogram of the separation distance is obtained, and the potential energy profile can be calculated by assuming the histogram represents a Boltzmann distribution. In order to test TIRM as a quantitative tool for measuring colloidal forces, a known force, gravity, was measured. Polystyrene spheres having a diameter between 7 and 15 jam were studied in aqueous solutions at elevations predicted to be between 80 and 120 nm above the plate. Thus, gravitational forces corresponding to masses as small as 10 pg were determined with an rms error of 6%. While absolute separation distances cannot yet be determined, changes in separation as small as 1 nm can be detected. Although the scattering intensity is much less sensitive to lateral motion of the particle than to motion normal to the plate, lateral motion can distort the potential energy profile and, therefore, must be minimized or taken into account. © 1990, American Chemical Society. All rights reserved.